Integrated Metabolomics and Transcriptome Analyses Unveil Pathways Involved in Sugar Content and Rind Color of Two Sugarcane Varieties

Abstract

Metabolic composition can have potential impact on several vital agronomic traits, and metabolomics, which represents the bioactive compounds in plant tissues, is widely considered as a powerful approach for linking phenotype-genotype interactions. However, metabolites related to cane traits such as sugar content, rind color, and texture differences in different sugarcane cultivars using metabolome integrated with transcriptome remain largely inconclusive. In this study, metabolome integrated with transcriptome analyses were performed to identify and quantify metabolites composition, and have better insight into the molecular mechanisms underpinning the different cane traits, namely, brix, rind color, and textures in the stems (S) and leaves (L) of sugarcane varieties FN41 and 165402. We also identified metabolites and associated genes in the phenylpropanoid and flavonoid biosynthesis pathways, starch and sucrose metabolism. A total of 512 metabolites from 11 classes, with the vast majority (122) belonging to flavonoids were identified. Moreover, the relatively high amount of D-fructose 6-p, D-glucose6-p and glucose1-p detected in FN41L may have been transported and distributed by source and sink of the cane, and a majority of them reached the stem of sugarcane FN41L, thereby promoting the high accumulation of sugar in FN41S. Observations also revealed that genes such as C4H, CHS, F3H, F3'H, DFR, and FG2 in phenylpropanoid and flavonoid biosynthesis pathways were the major factors impacting the rind color and contrasting texture of FN41 and 165204. Further analysis revealed that weighted gene co-expression network analysis (WGCNA) hub genes and six transcription factors, namely, Tify and NAC, MYB-related, C2C2-Dof, WRKY, and bHLH play a key role in phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, starch and sucrose metabolism. Additionally, metabolites such as L-phenylalanine, tyrosine, sinapaldehyde, pinobanksin, kaempferin, and nictoflorin were the potential drivers of phenotypic differences. Our finding also demonstrated that genes and metabolites in the starch and sucrose metabolism had a significant effect on cane sugar content. Overall, this study provided valuable insight into the molecular mechanisms underpinning high sugar accumulation and rind color in sugarcane, which we believe is important for future sugarcane breeding programs and the selection of high biomass varieties.

Keywords: WGCNA; flavonoids; metabolome and transcriptome; sugar metabolism; sugarcane.

FIGURE 1

Physiological characteristics of FN41 and 165204 sugarcane cultivars, bar = 1 cm (A); Comparison of agronomic traits between FN41 and 165204 (B). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Principal component analysis (PCA) of the metabolites detected in the sugarcane stem and leaf samples with three biological replicates. “FN41L” and “165204L” represent the leaf tissues of sugarcane varieties FN41 and 165204, respectively; “FN41S” and “165204S” represent the stem tissues of sugarcane varieties FN41 and 165204, respectively, similarly hereinafter.

FIGURE 3

Venn diagrams illustrating differential metabolism between two varieties in the same compartments (A), and in the stems and leaves of the same variety (B).

FIGURE 4

Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEMs between (A) 165204S_vs_FN41S, (B) FN41S_vs_FN41L, (C) 165204L_vs_FN41L, and (D) 165204S_vs_165204L.

FIGURE 5

Venn diagram of the DEGs of the four comparison groups.

FIGURE 6

RT-qPCR analysis showing the significance level of various genes identified in the different tissues of cane.

FIGURE 7

Co-expression network analysis. (A) Hierarchical cluster tree showing 20 modules obtained by weighted gene co-expression network analysis (WGCNA). The gray modules represent genes that are not divided into specific modules. Each branch in the tree points to a gene. (B) Matrix of module-metabolite associations. Combining the gene expression profile data of stem and leaf tissues of different sugarcane varieties and the change patterns of phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, starch and sucrose metabolism, displayed by the WGCNA analysis. The number of genes in each module is shown in the left box, followed by correlation coefficient and p-value between modules and metabolites, which are displayed at the intersection of rows and columns. (C) Co-expression sub-network analysis of purple, pink, and yellow modules related to the accumulation of phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, starch and sucrose metabolism. The first 50 nodes of purple, pink, and yellow modules to build the network were selected, and transcription factors are shown in red.

FIGURE 8

Diagram of phenylpropanoid and flavonoid biosynthesis pathways with their related DEGs and DEMs. PAL, phenylalanine ammonia lyase; PTAL, phenylalanine/tyrosine ammonia-lyase; C4H, cinnamate4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3H, P-coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CHI, chalcone isomerase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; F5H, ferulate5-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; POD, peroxidase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; Non-significant DEGs are shown in black. The solid line indicates the metabolic reactions in only one step. The dash line presents more than one step of the metabolic reaction.

FIGURE 9

Co-expression analysis illustrating genes and metabolites in phenylpropanoid and flavonoid biosynthesis pathway. Nodes represent genes or metabolites, and edges represent relationships between any two genes. Edges with solid and dashed lines represent positive and negative correlations, respectively, as determined by a Pearson correlation coefficient > 0.8 or < –0.8, respectively.

FIGURE 10

Diagram of starch and sucrose metabolism pathway with their related DEGs and DEMs. SPS, sucrose phosphate synthase; SPS, sucrose phosphatase; SUS, sucrose synthase; INV, invertase; HK, hexokinase; scrK, phosphofructokinase; non-significant DEGs are shown in black. The solid line indicates the metabolic reactions in only one step. The dash line presents more than one step of the metabolic reaction.